MIC661 Molecular Diagnostics Lecture 1 (Oct 2024) PDF

MIC661 Molecular Diagnostics TOPIC 1: INTRODUCTION By Dr. Muhd Hanis bin Md Idris  Table of Contents 01 02 03 Course Info Study Plan Topic 1 Course description Time table...

MIC661 Molecular Diagnostics TOPIC 1: INTRODUCTION By Dr. Muhd Hanis bin Md Idris  Table of Contents 01 02 03 Course Info Study Plan Topic 1 Course description Time table Terminologies in Course learning Attendance Topic & molecular diagnostics outcome Course syllabus assessment  01 Course Info Course Description This course is about the applications of molecular methods in the diagnosis of infectious diseases, epidemiologic investigations and infection control in both humans and plants. The use of nucleic acid amplification-based methods in detecting and identifying pathogens from clinical and plant materials will be highlighted. The molecular methods for chromosomal and plasmid DNA profiling, DNA fingerprinting or restriction fragment-length polymorphism analysis, detection of genes or gene mutations responsible for drug resistance, detection of virulence genes, molecular strain typing or genotyping that are frequently used in epidemiology analysis and infection control will also be covered. Emerging technologies including mass spectrometry and sequencing-based pathogen identification, characterization and screening, as well as other novel and late-breaking approaches will also be introduced.  Course Learning Outcome Explain the principle and methods of DNA hybridization, polymerase chain reaction (PCR) and genome sequencing techniques and their applications in diagnosis of infectious diseases and epidemiology analysis and infection control in both humans and plants (C2) Elaborate and review current molecular diagnostic techniques in the detection and the identification of pathogens (C5) Manipulate results of laboratory experiments and report the interpretation in a written form (P4)  02 Study Plan  Study Plan Important Dates Timetable Entrance Survey (07/10/24-03/11/24) Please check the day and time Exit Survey (06/01/25 – 09/02/25) and respond SuFO (06/01/25 – 09/02/25) Class Representative Test 1 (11/11/24) F2F/ODL platform (Microsoft Team) Final Exam (Feb 2025) Attendance (Manual/uFuture) – 80% Topic & Syllabus Assessment 7 main topics to covers Lab report - 25% 14 weeks to go Test - 25% 3 hours credit Final exam - 50% 120 hours SLT  ODL Platform Microsoft Team > MIC661 Molecular https://bit.ly/3YpXIys Diagnostics (Oct 2024) Entrance Survey Status (16/38)  03 Topic 1 Introduction to Molecular Diagnostics  Topic 1: Introduction to Molecular Diagnostics 1.1 What is molecular diagnostics? 1.2 Why molecular diagnostics? 1.2.1 Specificity and sensitivity 1.2.2 Personalized health medicine 1.3 Terminologies in molecular diagnostics What is molecular diagnostics? Molecular diagnostics (MDx) tests typically analyze key DNA, RNA, or protein biomarkers (analytes) to identify a disease, determine its course, evaluate response to therapy, or predict individual predisposition to a disease. The techniques applied involve analysis of DNA sequences, DNA methylation patterns, gene expression profiles, protein expression, or combinations of these biomarkers. Such biomarkers provide direct information about genotypic and/or phenotypic changes associated with specific diseases or responses to treatment. Biomarker analysis has also become an important tool in drug discovery, preclinical drug development, and patient monitoring during clinical trials Why molecular diagnostics is used? Specificity Sensitivity Personalized medicine Molecular diagnostics is the fastest growing segment of the broader diagnostics market USD 20,000,000,000 The market for molecular diagnostics is expected to grow from $8.5 billion at present to around $20 billion by 2030 Specificity Specificity = the ability of a diagnostic test to correctly identify individuals who do not have a particular disease or condition. A specific test will correctly identify most, if not all, individuals who do not have the disease, with a low rate of false-positive results. For example, in the case of a molecular diagnostic test for a particular pathogen, a test with high specificity would correctly identify individuals who are not infected with the pathogen as negative. This is important because a test with low specificity could result in false-positive results, leading to unnecessary treatment and unnecessary costs. The specificity of a diagnostic test is typically expressed as a percentage, indicating the proportion of individuals without the disease who test negative for it. For example, a test with a specificity of 95% would correctly identify 95 out of 100 individuals who do not have the disease, with 5 false- positive results. Sensitivity Sensitivity = the ability of a diagnostic test to correctly identify individuals who have a particular disease or condition. A sensitive test will correctly identify most, if not all, individuals who have the disease, with a low rate of false-negative results. For example, in the case of a molecular diagnostic test for a particular pathogen, a test with high sensitivity would detect the presence of the pathogen in almost all individuals who are infected with it. This is important because a test with low sensitivity could result in false-negative results, leading to missed diagnoses and delays in treatment. The sensitivity of a diagnostic test is typically expressed as a percentage, indicating the proportion of individuals with the disease who test positive for it. For example, a test with a sensitivity of 95% would correctly identify 95 out of 100 individuals who have the disease, with 5 false-negative results. Specificity and Sensitivity Specificity and sensitivity of DNA based tests are much higher, and the test is quicker than identification by traditional laboratory methods. In the case of human immunodeficiency virus (HIV), the initial detection limit in the late 1980s was 22 days post-infection. However, the advent of nucleic acid testing for HIV RNA shortened the time to 11 days post- infection. This highlights another distinct advantage offered by molecular diagnostics over immunoassays–amplification. The polymerase chain reaction (PCR) allows for the billion-fold amplification of available genetic material, thereby allowing the detection of minute amounts of genetic material and greatly increasing the sensitivity of the assay. No such amplification technique exists for proteins. Pros/Cons of Molecular Diagnostics Advantages Disadvantages Reduce the number of false-negative Expensive tests Require proper specimen Decrease in turn-around-times collection time, handling Fast and accurate - permits large-scale conditions, storage temperature screening of organisms and transportation Reduce the chance of cross Require sophisticated equipment contamination Test inhibition Decrease this health risk for the Lack of validation laboratory workers  Personalized medicine An approach to healthcare that uses information about an individual's unique genetic, environmental, and lifestyle factors to tailor medical treatment and disease prevention strategies. The goal of personalized medicine is to provide the right treatment to the right patient at the right time, resulting in better outcomes and reduced healthcare costs. Personalized medicine relies on the use of advanced technologies, such as genomic sequencing, proteomics, and metabolomics, to identify individual variations in genes, proteins, and other molecules that can affect disease risk, progression, and response to treatment. By analyzing this information, healthcare providers can develop targeted interventions that are tailored to each patient's specific needs, improving the effectiveness and safety of treatment.  Personalized medicine Recognize that patients differ on a molecular level in ways that have a major impact on therapy effectiveness. 01 02 03 Predictive Participatory Preventive Using information Providing patients with Helping the patient to gleaned from a patient's actionable information take preventative data to anticipate the that they can utilize to actions based on the conditions that they are take an active role in risk factors identified most likely to develop. their treatment and improve it  Precision medicine Classify patients with a disease into subgroups, considering their phenotypic findings, such as biomarkers or genomics. Sarvan & Nori (2021)  The applications and technology Molecular diagnostics (MDx) has traditionally been utilised in life science research, infectious disease testing, and cancer diagnostics, but the market for MDx is expanding into new fields like companion diagnostics, liquid biopsy, direct-to-consumer, and more. Debnath et al. (2010). Molecular Diagnostics: Promises and Possibilities  Technologies such as PCR, serological assays and rapid diagnostics help us understand the spread of COVID-19. But how do they do that? https://youtu.be/824LMTRrYqM 1. Fluorescence 2. 3. 4. 5. Pathogen PCR: Polymerase Real-time Multiplex detection: chain inAsitu PCR reaction hybridization The technique identification (PCR):that A (FISH): method allows of A the hybridization the ofcausative nucleic simultaneous detection acid agent technique ofamplification amplified detection of an that infection, DNA of uses that multiple orcan Terminologies in Molecular Diagnostics fluorescent such RNAdetect as in pathogens real-time, a bacterium, the probes presence inenabling atosingle virus, target of specific reaction. the fungus, specific quantification DNA or DNA parasite. ororRNA RNA ofsequences the sequences target in molecule ina situ sample. (i.e., in ain sample. the original tissue or sample). 1. Pathogen detection: The identification of the causative agent of an infection, such as a bacterium, virus, fungus, or parasite. 2. Polymerase chain reaction (PCR): A method of nucleic acid amplification that can detect the presence of specific DNA or RNA sequences in a sample. 3. Real-time PCR: A PCR technique that allows the detection of amplified DNA or RNA in real-time, enabling the quantification of the target molecule in a sample. 4. Multiplex PCR: A PCR technique that allows the simultaneous detection of multiple pathogens in a single reaction. 5. Fluorescence in situ hybridization (FISH): A hybridization technique that uses fluorescent probes to target specific DNA or RNA sequences in situ (i.e., in the original tissue or sample). 6. Antibiotic 7. 8. 9. 10.Next-generation Microarray: Viral Genome load:resistance: sequencing: The A technology amount sequencing The The of ability viral that process (NGS): allows DNA of bacteria of or A the high-throughput determining RNAsimultaneous present to resistthe in the asequencing complete detection patient's effects ofblood DNA and antibiotics. technology analysis sequence or other Terminologies in Molecular Diagnostics Molecular of that bodily ofmultiple an canorganism's fluids. generate diagnostics DNAThis ormillions is genome. RNAan cansequences important help ofThis DNA identify can indicator or inRNA help aantibiotic-resistant sample. sequences identify of the severity unique in a single strains genetic of a viral run, offeatures bacteria, infection enabling of enabling and athe pathogen identification the effectiveness morethat effective may of a be wide oftreatment antiviral important range treatment. ofand for pathogens diagnosis reducinginthe oratreatment. sample. spread of antibiotic resistance. 6. Next-generation sequencing (NGS): A high-throughput sequencing technology that can generate millions of DNA or RNA sequences in a single run, enabling the identification of a wide range of pathogens in a sample. 7. Microarray: A technology that allows the simultaneous detection and analysis of multiple DNA or RNA sequences in a sample. 8. Viral load: The amount of viral DNA or RNA present in a patient's blood or other bodily fluids. This is an important indicator of the severity of a viral infection and the effectiveness of antiviral treatment. 9. Antibiotic resistance: The ability of bacteria to resist the effects of antibiotics. Molecular diagnostics can help identify antibiotic-resistant strains of bacteria, enabling more effective treatment and reducing the spread of antibiotic resistance. 10. Genome sequencing: The process of determining the complete DNA sequence of an organism's genome. This can help identify unique genetic features of a pathogen that may be important for diagnosis or treatment. Terminologies in Molecular Diagnostics Glossary and representation of terms related to diagnostics test Glossary and representation of terms related to diagnostics test References Glossary and representation of terms related to diagnostic tests. March 2016 –Version 01. http://www.orpha.net/orphacom/cahiers/docs/GB/Glossary_terms_related _to_diagnostic_tests.pdf Seith, H. and Schumacher, S. (2013). Molecular Diagnostics. Springer. Cagle, P. T. and Allen, T.C. (2009). Basic Concepts of Molecular pathology. Springer. Debnath, M. et al. (2010). Molecular Diagnostics: Promises and Possibilities. Springer.  Thanks! Does anyone have any questions? [email protected] +60 16 666 4359 CREDITS: This presentation template was created by Slidesgo, including icons by Flaticon, and infographics & Slidesgo Flaticon images by Freepik and illustrations by Stories Freepik Stories Please keep this slide for attribution